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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, however with following deficiencies: hematology and clinical chemistry examinations were only conducted for males and no neurobehavioural examinations were done.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1996
Deviations:
yes
Remarks:
no neurobehavioural examination performed; hematology and clinical chemistry only was done on males.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain as given in study report: Sprague-Dawley; Crj:CD(SD)
- Source: Charles River Laboratories Japan, Inc., Atsugi, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 354 - 386 g (males), 216 - 241 g (females)
- Housing: stainless steel cage
- Diet: NMF from Oriental Yeast Co., Ltd, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (light intensity 150 - 300 lux)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in corn oil. Dosing solutions were prepared once or more a week, kept under seal, cool and dark until use and used within 7 days after preparation. 5 (w/v)% was confirmed to be stable for at least 8 days under cool and dark condition.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After 18 days of pregnancy, females were caged in stainless cage with a nursing tray and nesting stuff (alfa dry).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the concentration was conducted using a sample by random-choice from each batch. Since 98.6 - 104% was shown as a result, it was confirmed that given amount of test item was included.
Duration of treatment / exposure:
Females: 14 days before mating until day 3 of lactation (41 - 55 days)
Males: 45 days
Frequency of treatment:
daily
Details on study schedule:
- Terminal sacrifice was on day 45 for males and on day 4 of lactation for females.
- Dose selection rationale: A preliminary test was performed to determine the dose range for the main test. Groups of male and female animals received 0, 250, 500, 750 and 1000 mg/kg bw/day of the test item for 14 consecutive days. No mortality was observed and the general health state was not affected by treatment; no changes in weight, food consumption, gross pathology, organ weights, blood chemistry and hematology were noticed.
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males on days 1, 8, 15, 22, 29, 36 and 43 of administration; Females on days 1, 8 and 15 before mating, on days 0, 7, 14 and 21 of gestation and on days 0 and 4 of Lactation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: days 1-8, 8-15, 22-29, 29-36, 36-43 and 43-45 in male animals; days 1-8, 8-15 before mating, days 0-7, 7-14 and 14-21 of gestation and during days 0-4 of lactation in females.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (16 hours)
- How many animals: 12 males per dose
- Parameters checked: Hematocrit (Hct), Hemoglobin (Hgb), Red Blood Cell Count (RBC), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), White Blood Cell Count (WBC), Differential leukocyte counts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Animals fasted: Yes (16 hours)
- How many animals: 12 males per dose
- Parameters checked: Total protein, albumin, A/G, glucose, Blood Urea Nitrogen (BUN), creatinine, total cholesterol, Glutamate‐Oxaloacetate Transaminase (GOT), Glutamate–Pyruvate Transaminase (GPT), gamma-GTP, total bilirubin, potassium, chloride, calcium, inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Estrous cyclicity (parental animals):
examined
Sperm parameters (parental animals):
not examined
Litter observations:
PARAMETERS EXAMINED (F1 OFFSPRING):
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain.
Postmortem examinations (parental animals):
ORGAN WEIGHTS: Yes, absolute and relative weights
- How many animals: 10- 12 animals per dose
- Organs: thymus, liver, kidneys, testes, epididymides, ovaries, lung.

GROSS PATHOLOGY: Yes
Males: thymus, lungs, liver, kidney, brain, heart, spleen, adrenal gland, seminal vesicles, prostate, testis and epididymides.
Females in control and highest dose group at day 4 of lactation: thymus, lung, liver, kidney, ovary, heart, spleen, adrenal, colon.
Non-pregnant females: skin, breast, lymph nodes, salivary glands, sternum, femur (including marrow), thymus, trachea, bronchi and lungs, heart, thyroid and parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, colon, liver, pancreas, spleen, kidney, adrenal gland, bladder, ovaries, uterus, vagina, eye, Harderian gland, brain, pituitary, spinal cord.
Females with all dead pups: skin, breast, lymph nodes, salivary glands, sternum, femur (including marrow), thymus, trachea, bronchi, lungs, heart, thyroid and parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, colon, liver, pancreas, spleen, kidney, adrenal gland, bladder, ovaries, uterus, vagina, eye, Harderian gland, brain, pituitary and spinal cord.
Newborns: major organs.

HISTOPATHOLOGY: Yes
Males in control and highest dose group with proven fertility: brain, thymus, heart, lungs, liver, kidney, spleen, testis and adrenal gland.
Females in control and highest dose group at day 4 of lactation: brain, thymus, heart, lungs, liver, kidney, spleen, adrenal and ovaries
Non-pregnant females and infertile males: brain, thymus, heart, lungs, liver, kidney, spleen, adrenal glands, vagina, uterus, ovaries, testis, epididymides, seminal vesicles, prostate and pituitary gland.
Females with all dead pups: skin, breast, lymph nodes, salivary glands, sternum, femur (including marrow), thymus, trachea, bronchi and lungs, heart, thyroid and parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenal gland, bladder, ovaries, uterus, vagina, eye, Harderian gland, brain, spinal cord and pituitary gland.
Postmortem examinations (offspring):
SACRIFICE
- Four days after birth

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
- Performed but detail information was not stated.
Statistics:
Barlett test, One-way ANOVA, Kruskal-Wallis test, Dunnett test, Scheffe test, chi-square test
Reproductive indices:
copulation index, fertility index, gestation index, implantation index, delivery index
Offspring viability indices:
live birth index, viability index on day 4, type and incidence of external anomalies

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality was not observed in either group throughout the treatment period for both sexes.
Fracture of incisors was seen in one control male (day 5 to 7), and in the 250 mg/kg bw/day group, two males showed alopecia and crusting; the findings were regarded as not treatment-related. The females were inconspicuous.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Difference in body weight between treatment groups and the control group throughout the treatment period was not observed in both sexes. In males, higher mean food consumption was observed in the 250 and 500 mg/kg bw/day groups compared with control; however, the finding was not considered to be a relevant effect. In females, no difference in food consumption between treated and and control animals was noticed

ESTROUS CYCLE (PARENTAL ANIMALS)
No difference in estrous cycle was observed for the females between treated and control.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance displayed no significant changes between treatment groups and controls (estrous cycle, copulation index, fertility index, gestation length, gestation index, delivery conditions, or implantation index). In control group and 1000 mg/kg bw/day, since there was bias of the number of male and female infants, sex ratio was defference between in these groups. Since many pups died before day four of lactation in the control group, number of total live female pups were significant different between the control group and 1000 mg/kg bw/day group. But no difference was found in delivery, pregnant duration, the number of corpora lutea, number of implantation scars, and fetal birth of live or dead pups, the birth rate, implantation rate, delivery percentage and fertility rate.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males of the 1000 mg/kg bw/day group, actual weight of the thymus was increased compared to control group. But this change was regarded as non-relevant effect because no such difference was observed in females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In fertile males, few cases of colored spots / areas in lung were reported (control and treated animals) ; the findings were nor related to treatment. Also red spots / areas in the thymus were reported for 2 males of the 500 mg/kg bw/day group, and gray spots / areas of the skin wereobserved in one male of the 250 mg/kg bw/day group.
In naturally delivering females and in each group including control, thymus atrophy, colored spots / area in the lungs, atrophy of the large intestine, white spots / areas in the liver, cysts of the ovary and scarring of the kidney were reported; all these changes were without toxicological relevance. Two animals in 250 mg/kg bw/day group were not successfully pregnant but showed no abnormal findings. The two animals, whose all pups died before day 4 of lactation, were control animals and one of them showed redness of the eye.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In the fertile males, granuloma of the liver lesion, basophilic tubules of the kidney, acidophilic bodies appearance in the kidney and vacuolated adrenal glands were observed. No abnormality was observed in thymus in the 1000 mg/kg bw/day group, where increase of organ weights was observed. In females having delivered naturally, pigmented spleen, aggregated macrophages in the lungs, focal granulomas in the liver, basophilic tubules and vacuolation of the kidney were observed in each group. In the two females of the control group with pups having died before day 4 of lactation, mammary gland hyperplasia, accumulations of macrophages, blood vessel expansion arteritis of the uterus were observed. An infertile male in 250 mg/kg bw/day group showed sperm granulomas of the epididymides.
However, all these changes were considered to be not toxicologically relevant, since there were no difference of the number of occurrence between each group.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(reproduction)
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on estrous cycle, copulation index, fertility index, gestation length, number of corpora lutea or implantation, implantation index, delivery index or parturition.
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects (clinical signs; mortality; body weight; food consumption; hematology; clinical chemistry; gross pathology; organ weights; histopathology) observed in all dose groups.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
no difference was observed between pups from treated groups and from control.

CLINICAL SIGNS (OFFSPRING)
2 case of hypodermic hemorrhage, 1 case of imperforate anus and 1 case of rudiment tail were observed in control group. 1 case of nanooffspring was seen in the 250 mg/kg bw/das group and 1 case of short tail was seen in the 500 mg/kg bw/day group.

BODY WEIGHT (OFFSPRING)
Regarding weight, no difference between treated and control groups was observed.

SEXUAL MATURATION (OFFSPRING)
not determined.

ORGAN WEIGHTS (OFFSPRING)
not determined.

GROSS PATHOLOGY (OFFSPRING)
At Necropsy, thymic remnant in the neck was observed in the control group and at 250 and 1000 mg/kg bw/day, diaphragmatic hernia was observed in two dead pups at 500 mg/kg bw/day.

HISTOPATHOLOGY (OFFSPRING)
no data.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on live birth index and viability index on day 4, no abnormalities.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1:

Summary of reproductive performance in rats treated orally with Methyl dodecanoate in the combined repeated dose and reproductive/developmental toxicity screening test

Dose level

0 mg/kg

250 mg/kg

500 mg/kg

1000 mg/kg

No. of pairs mated

12

12

12

12

No. of pairs copulated

12

12

12

12

No. of pregnant females

12

10

12

12

Copulation index (%)

100 %

100 %

100 %

100 %

Fertility index (%)

100 %

83.3 %

100 %

100 %

Estrus cycle (days, mean)

4.5

4.6

4.5

4.7

 

Table 2

Findings of delivery in dams treated orally with Methyl dodecanoate and observations on their pups (F1) in the combined repeated dose and reproductive/developmental toxicity screening test

Dose level

0 mg/kg

250 mg/kg

500 mg/kg

1000 mg/kg

No. of dams observed

12

10

12

12

No. of dams delivering live newborns

12

10

12

12

Duration of gestation (mean)

22.9

22.6

22.6

22.4

No. of corpora lutea (mean)

221 (18.4)

168 (16.8)

204 (17.0)

209 (17.4)

No. of total implants (mean)

188 (15.7)

161 (16.1)

173 (14.4)

180 (15.0)

No. of total pups born (mean)

171 ( 14.3)

147 (14.7)

157 (13.1)

163 (13.6)

No. of total live pups born (mean)

170 (14.2)

146 (14.6)

156 (13.0)

163 (13.6)

No. of total live pups on day 4

 

 

 

 

  Male

84 (7.0)

72 (7.2)

71 (5.9)

64 (5.39

  Female

57 (4.8)

61 (6.1)

81 (7.4)

97 (8.1)*

No. of total dead pups born

1 (0.1)

1 (0.1)

1 (0.1)

0 (0.0)

     stillbirth

0 (0.0)

1 (0.1)

1 (0.1)

0 (0.0)

cannibalism

1 (0.1)

0 (0.0)

0 (0.0)

0 (0.0)

Gestation index (%)

100 %

100 %

100 %

100 %

Implantation index (%)

84.0

95.9

85.2

86.7

Delivery index (%)

89.8

91.2

88.9

90.6

Live birth index (%)

99.5

99.4

99.5

100.0

Viability index on day 4

 

 

 

 

  Male

76.7

91.3

97.8

95.1

  Female

78.2

94.5

97.5

100.0

* Significantly different from control p < 0.05

 

 

Applicant's summary and conclusion